Progresses in gene recombinant technology have enabled the large scale production of physiologically active proteins such as monoclonal antibodies for diagnostic and therapeutic applications. The recent development of humanized monoclonal antibodies has spawned an unprecedented interest in using these molecules as therapeutics since they can specifically target disease implicated molecules, thus essentially side-stepping the secondary effects that are usually associated with conventional drug therapies. Therapeutic monoclonal antibodies have thus become the fastest growing area of biopharmaceutical applications.
While the production and purification scales have reached industrial levels, there are novel concerns about strategies for their stable formulation and delivery. Physical and chemical instability of antibodies is really a complex function of solution conditions and temperature. Antibodies are for example susceptible to deamidation, isomerization, oxidation, proteolysis, aggregation and other covalent modifications. However, unlike other model proteins, antibody stability is not necessarily dependent on protein concentration, buffer concentration, salt concentration, or agitation. Some antibodies can be stabilized by changing different parameters in the formulation, like pH, excipients and buffer, though. However, antibody stabilization is a hard task as their activity is very sensitive to environment conditions which render it very difficult to predict, notably because each antibody has a very specific and characteristic stability profile. The lack of effect for primary factors commonly known to affect physical stability suggests that the mechanism of antibody stability is counter intuitive and unlike other proteins. Further, those aggregation phenomena are suspected to result in potential clinical side-effects or toxicity since aggregates can reduce the efficacy and enhance the immunogenicity of the protein drug (Demeule et al., 2006, Eur. J. Pharm. Biopharm., 62:121-30).
Antibody aggregation is also a source of batch to batch variabilities in the antibody production chain and its control leads to regulatory and quality control burden which have extremely costly consequences.
Further, aggregation propensity of antibodies affects their stability in storage, including shelf-life and their useable administration time, once removed from optimum storage conditions.
Bevacizumab (Avastin®) is a recombinant monoclonal humanized IgG1 antibody with a molecular weight of 149 kDa that binds to and inhibits the biologic activity of vascular endothelial growth factor (VEGF) which has been approved for the treatment of metastatic cancer of the colon or rectum. Currently, bevacizumab is widely used off-label for the treatment of neovascular age-related macular degeneration (AMD), a common form of progressive age-related vision loss (Andreoli et al., 2007, Curr. Opin. Opthalmol., 18:502-8), by intraocular injection. This off-label use was introduced after Ranibizumab (Lucentis®), a humanized monoclonal antibody fragment derived from the same murine antibody precursor as bevacizumab with a molecular weight of 48 kiloDalton (kDa) had been registered in 2006 for the treatment of AMD. For ranibizumab, a monthly injection into the vitreous cavity is recommended to maintain therapeutically effective drug concentrations (Regillo et al., 2008, Am. J. Opthalmol., 145, 239-48) and the same frequency is generally reported for bevacizumab injections. Nevertheless, a reduced frequency of injections would be favourable because of patient discomfort and risk of complications (Brown et al., 2007, Am. J. Opthalmol., 144:627-37).
Several clinical trials have reported the administration of both bevacizumab and some anti-inflammatory drugs such as triamcinolone acetonide or dexamethasone in the treatment of neovascular age-related macular degeneration through separate intravitreal injections of the two drugs in order to address the multifactorial pathogenesis of AMD (Augustin et al., 2007, Retina, 27, 133-40; Ahmadieh et al., 2008, Graefes Arch. Clin. Exp. Opthalmol., 246(4):483-9; Colucciello et al., 2008, J. Ocul. Pharmacol. Ther., February; 24(1):15-24).
Among one of the advantages that promoted the off-label use of bevacizumab is its much lower price as compared to its fragment ranibizumab that lacks an Fc portion. However, bevacizumab, which is an intact antibody, presents, as such, the significant aggregation propensity commonly found for antibodies mentioned above. Surface active agents like polysorbate 20 and 80 which are widely used to reduce the rate of protein aggregation are also used in the formulation of AVASTIN where 0.04% of polysorbate 20 is present. However, surface active agents can only be used in antibodies formulations at percentages of 0.01% to 0.05% because of their participation in protein unfolding at higher concentrations.
Since aggregation is a major issue for the production, formulation and/or stability of therapeutic antibodies since antibody aggregates can lead to loss of biological activity, loss of solubility and even increased immunogenicity the development of a method of preparation and/or formulations of antibodies that would lead to a longer shelf-life and stability of those antibodies would be highly desirable.